We have cloned and sequenced the genome of the H strain of HCV, and constructed a putative full length cDNA clone of the genome. We have evaluated that clone in cell culture systems as well as by direct transfection of RNA transcripts of this clone into chimpanzee liver. While this original clone is not infectious, in collaboration with C.M. Rice, Washington U. St Louis, significant new information has been developed that gives us reason to believe that we now have a full length clone which is about to be tested chimpanzees. In an in vitro cell culture systems continue to be investigated. Both a liver cell line, THLE5b and lymphocyte cell lines are being studied and low level replication of HCV replication has been demonstrated by PCR. Novel assay systems for viral replication are being developed based on the in vivo detection of HCV specific enzymes. The HCV intracellular RNA dependent RNA polymerase and the NS3 protease can be detectected by expression of the specific substrates for these enzymes within the cell in systems designed to detect the reaction product with high sensitivity. These culture systems can be applied to the problem of viral neutralization as well as studies of viral replication. Both the polymerase and the protease assays have the potential to be very useful for screening antiviral drugs and biologics. Though this system remains difficult to work with it still provides a means to study neutralizing anti- bodies without the use of the animal (chimpanzee) model.It is hoped that this system can be further developed to a practical system for studying viral replication. Earlier work that defined the 2 viral proteases of HCV is now being applied by several labs attempting to develop specific therapeutics for HCV. Understanding the viroplogy and molecular biology of HCV is vitally important for mechanism of action of therapeutics. We may also be able to develop direct in vitro assays for standardization and potency testing therapeutics of both the biologic and drug classes. In vitro culture systems and infectious cDNA/RNA will be invaluable tools for both the developement and evaluation of diagnostics, therapeutics and vaccines against HCV as well as a tool for studying basic viral functions that could then be targeted by antivirals or vaccines.